Expression of IL-34 correlates with macrophage infiltration and prognosis of diffuse large B-cell lymphoma

Abstract

Objectives: Infiltration of macrophages through the tyrosine kinase receptor CSF1R is a poor prognosis factor in various solid tumors. Indeed, these tumors produce CSF1R ligand, macrophage colony-stimulating factor (M-CSF) or interleukin-34 (IL-34). However, the significance of these cytokines, particularly, the newly discovered IL-34 in haematological malignancies, is not fully understood. We therefore analysed the role of IL-34 in diffuse large B-cell lymphoma (DLBCL), the most common subtype of malignant lymphoma.

Methods: We analysed formalin-fixed paraffin-embedded lymphoma tissues of 135 DLBCL patients for the expression of IL-34 and the number of macrophages, and the survival of these patients. The expression of IL-34 in DLBCL cell lines and the activity of IL-34 to induce the migration of monocytic cells were also characterised.

Results: Several lymphoma tissues showed a clear IL-34 signal, and such signal was detectable in 36% of patients. DLBCL cell lines also expressed IL-34. Interestingly, the percentage of IL-34⁺ patients in the activated B-cell subtype was significantly higher than that in the germinal centre B-cell subtype. More interestingly, IL-34⁺ patients showed shorter survival periods and higher number of macrophages in lymphoma tissues. The recruitment of monocytes is likely the first step for the higher macrophage density in the IL-34⁺ lymphoma tissues. Indeed, IL-34 induced the migration of monocytic cells.

Conclusion: Our results raise the possibility that IL-34 in lymphoma tissues of DLBCL patients recruits monocytes, leading to the higher number of macrophages in the tissues and poor prognosis of patients. IL-34 may be an additional therapeutic target of DLBCL.

Keywords: CSF1R; DLBCL; IL‐34; Macrophages; M‐CSF.

Figure 1

The expression of IL‐34 in the lymphoma tissues of in diffuse large B‐cell lymphoma (DLBCL) patients. (a) The skin sample from healthy donor was stained for IL‐34 as a positive control (left) because IL‐34 is known to highly express in keratinocytes (indicated by arrows).5, 6 The lymph nodes showing reactive hyperplasia were also stained for IL‐34 as a negative control (right). (b) Representative immunostaining of IL‐34 in the lymphoma tissues of DLBCL patients is shown. The upper and lower panels are IL‐34⁻ and IL‐34⁺ cases, respectively, and both cases are GCB subtype.

Figure 2

The overall survival of in diffuse large B‐cell lymphoma (DLBCL) patients. The Kaplan–Meier survival analysis of DLBCL patients (n = 135) according to age (upper left), sex (lower left), the subtype of DLBCL (upper right) and IL‐34 expression (lower right) is shown.

Figure 3

The expression of IL‐34 in in diffuse large B‐cell lymphoma (DLBCL) cell lines. Daudi, SUDHL‐6, OCI‐Ly3 and U2932 cells were subjected to the real‐time RT‐PCR to quantify their mRNA level of IL‐34 (top), M‐CSF (middle) or CSF1R (bottom) followed by the normalisation to the mRNA level of GAPDH. The IL‐34‐, M‐CSF‐ or CSF1R mRNA level of DLBCL cell lines (SUDHL‐6, OCI‐Ly3 and U2932) was calculated relative to that of Daudi. Data are shown as the mean ± sd of three independent experiments. *P < 0.05.

Figure 4

The number of macrophages in the lymphoma tissues of in diffuse large B‐cell lymphoma (DLBCL) patients. (a) Representative immunostaining of Iba‐1⁺ macrophages (left) and CD163⁺ macrophages (right) in the lymphoma tissues of DLBCL patients is shown. The upper and lower panels are IL‐34‐negative and IL‐34‐positive cases, respectively, and both cases are GCB subtype. (b) The number (mm⁻²) of Iba‐1⁺ macrophages (left) and CD163⁺ macrophages (right) in the lymphoma tissues (n = 53) was compared between IL‐34⁺‐ and IL‐34⁻ groups. The randomly selected six tumor areas were counted for each specimen. The statistical significance of difference between groups was calculated using the Mann–Whitney U‐test.

Figure 5

The migration‐inducing activity of IL‐34. (a) In the upper panel, the migration of TF‐1‐fms cells towards rhIL‐34 or rhM‐CSF was measured using the transmigration chamber assay. rhIL‐34 or rhM‐CSF was added to the wells at the indicated concentrations, and cells were cultured for 18 h. The number of cells that migrated through the inserts was assessed using the MTT assay. In the lower panel, TF‐1‐fms cells were cultured for 3 days in the absence or presence of rhIL‐34 or rhM‐CSF at the indicated concentrations, and their growth was assessed using the MTT assay. Data are shown as the mean ± sd of three independent experiments. *P < 0.05. (b) The migration of TF‐1‐fms cells was assessed in the absence or presence of the CSF1R kinase inhibitor GW2580 (10 μm). rhIL‐34 or rhM‐CSF was used at 100 ng mL⁻¹. The cell migration is represented as percentages relative to that of the GW2580‐free cultures. Data are shown as the mean ± sd of three independent experiments. *P < 0.05. (c) The migration of TF‐1‐fms cells was assessed in the absence or presence of the indicated anti‐CSF1R monoclonal antibodies (10 μg mL⁻¹). rhIL‐34 was used at 100 ng mL⁻¹. The cell migration is represented as percentages relative to that of the antibody‐free cultures (left‐most). Data are shown as the mean ± sd of three independent experiments. *P < 0.05. (d) TF‐1‐fms cells were stimulated with 100 ng mL⁻¹ of rhIL‐34 or rhM‐CSF at 37°C for the indicated periods. Then, the cell surface expression of CSF1R was analysed by the flow cytometry, and their mean fluorescent intensity (MFI) value is represented as percentages relative to that of un‐stimulated cells. The level of cell surface CSF1R was constant over time under the cytokine‐free conditions (data not shown). Data shown are representative of four independent experiments with similar results. (e) TF‐1‐fms cells were stimulated with 100 ng mL⁻¹ of rhIL‐34 or rhM‐CSF at 37°C for 0.5 h (left, as a representative of CSF1R down‐regulation phase) or 12 h (right, as a representative of CSF1R recovery phase), and analysed as in d. Data are shown as the mean ± sd of four independent experiments. *P < 0.05.